PROJECT SUMMARY Until recently epithelial cells lining the mucosal tissues were not considered to be central in their role in innate immunity against invading pathogens, but have lately emerged as major cell types in the initial host defenses directed to colonizing microbes at various mucosal sites including oral mucosa. Our laboratory's recent studies focused on the innate oxidative stress responses of human gingival epithelial cells (GECs) to the oral pathogen, Porphyromonas gingivalis. This opportunistic organism successfully invades and persists in GECs leading to periodontal disease and is proposed to play specific roles in other severe chronic diseases such as rheumatoid arthritis, cancer, and diabetes thus affecting the U.S. population ranging from juveniles to geriatric age groups. Infection by P. gingivalis promotes a pro-survival phenotype and inhibits apoptosis in GECs mediated by a danger molecule, extracellular ATP (eATP). Interestingly, P. gingivalis, an effective colonizer of gingival mucosa is also able to modulate and suppress ROS generation by inhibiting the eATP/P2X7 receptor coupling which is linked to several key pathogen elimination host responses including NADPH oxidase (NOX) - induced ROS and anti-microbial autophagic pathway activation. NADPH oxidases specifically, although much better studied in professional phagocytes such as neutrophils, have never been characterized in GECs, and may provide critically novel insights into the molecular mechanisms of P. gingivalis' surivival and persistence in the oral cavity. Therefore, two Specific Aims are proposed in this study. Aim 1 will characterize early events in the host molecular defense mechanisms against intracellular bacteria through eATP-induced NOX multi- enzyme complex generated ROS in primary GECs. Aim 2 will define the NOX signaling processes and associated autophagic flux modulated in response to intracellular bacteria for successful persistence in primary GECs. Specifically, we will identify novel eATP-mediated NOX2-coupled ROS bacterial killing mechanisms in primary GECs during P. gingivalis infection. These will include studying myeloperoxidase activity and caspase 4/5-mediated lysosomal fusion of pathogen-containing phagosomes for degradation that P. gingivalis may control to assure persistence. Proposed studies will reveal key functions of epithelial cells as vital part of immune system and identify novel molecular circuitries closely orchestrated by P. gingivalis for successful colonization in gingiva. The insights gained from this study may translate to new therapeutic targets for controlling of P. gingivalis as well as other opportunistic pathogens and would therefore aid in development of highly targeted inhibition strategies to reduce the severity of chronic infections.